Biochemical and Molecular Action of Nutrients

An Extract of Lagerstroemia speciosa L. Has Insulin-Like Glucose

UptakeStimulatory and Adipocyte DifferentiationInhibitory
Activities in 3T3-L1 Cells1
Fang Liu,* Jae-kyung Kim,** Yunsheng Li,* Xue-qing Liu,** Jing Li* and Xiaozhuo Chen***2
*Edison Biotechnology Institute, Department of Biomedical Sciences, College of Osteopathic Medicine and
**Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701

KEY WORDS:

type II diabetes

banaba extract

insulin

Obesity is considered to be the most important risk factor

for noninsulin-dependent (type II) diabetes mellitus (NIDDM).3
NIDDM has been increasing at an alarming rate (projecting to
increase from 135 million in 1995 to 300 million worldwide in
2025), and has become a serious public health problem, particularly in developed countries (15). NIDDM is a complicated disease, involving both genetic and nongenetic factors
(57). Although the causes of NIDDM are not completely
known, obesity, hyperinsulinemia, hyperglycemia and insulin
resistance are closely associated with NIDDM (8 11). Insulin
plays multiple physiologic roles in the human body, including
the reduction of blood glucose levels and the promotion of
lipid biosynthesis in adipocytes (1214). It is highly desirable
to find antidiabetic agents that induce glucose uptake in

adipocyte differentiation

glucose uptake

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ABSTRACT The effects of extracts isolated from Lagerstroemia speciosa L. (banaba) on glucose transport and
adipocyte differentiation in 3T3-L1 cells were studied. Glucose uptakeinducing activity of banaba extract (BE) was
investigated in differentiated adipocytes using a radioactive assay, and the ability of BE to induce differentiation in
preadipocytes was examined by Northern and Western blot analyses. The hot water BE and the banaba methanol
eluent (BME) stimulated glucose uptake in 3T3-L1 adipocytes with an induction time and a dose-dependent
response similar to those of insulin. Furthermore, there were no additive or synergistic effects found between BE
and insulin on glucose uptake, and the glucose uptake activity of insulin could be reduced to basal levels by adding
increasing amounts of BE. Unlike insulin, BE did not induce adipocyte differentiation in the presence of 3-isobutyl1-methylxanthine (IBMX) and dexamethasone (DEX). BE inhibited the adipocyte differentiation induced by insulin
plus IBMX and DEX (IS-IBMX-DEX) of 3T3-L1 preadipocytes in a dose-dependent manner. The differences in the
glucose uptake and differentiation inhibitory activities between untreated cells and those treated with BE were
significant (P 0.01). The inhibitory activity was further demonstrated by drastic reductions of peroxisome
proliferator-activated receptor 2 (PPAR2) mRNA and glucose transporter-4 (GLUT4) protein in cells induced from
preadipocytes with IS-IBMX-DEX in the presence of BE. The unique combination of a glucose uptake stimulatory
activity, the absence of adipocyte differentiation activity and effective inhibition of adipocyte differentiation induced
by IS-IBMX-DEX in 3T3-L1 cells suggest that BE may be useful for prevention and treatment of hyperglycemia and
obesity in type II diabetics. J. Nutr. 131: 22422247, 2001.

cells, but, unlike insulin, do not simultaneously up-regulate

lipogenesis.
Leaves of the tropical plant Lagerstroemia speciosa L. (banaba in the Tagalog dialect in the Philippines) have been used
as a folk medicine for treatment of diabetes and kidney diseases. The extract from banaba significantly reduced blood
glucose and insulin levels in type II KK-Ay diabetic mice (15).
It was reported recently that the extract exhibited an antiadipogenic activity by effectively reducing weight gain and
parametrial adipose tissue in female diabetic mice (16). However, no study has reported the mechanisms of action by the
extract at the cellular and molecular levels, and it is not
known how the extract exerts these effects.
In this study, the ability of the banaba extract (BE) to
stimulate glucose uptake in 3T3-L1 adipocytes was examined
and compared with that of insulin. In addition, the effects of
BE on differentiation of preadipocytes into adipocytes, a process induced by an insulin/3-isobutyl-1-methylxanthine/dexamethasone (IS-IBMX-DEX) cocktail were also investigated.
The expression of peroxisome proliferator-activated receptor
2 (PPAR2), a nuclear protein that turns genes on and off
upon binding to molecules that belong to a group of compounds called peroxisome proliferators and is essential for
adipocyte differentiation (17,18), and of glucose transporter-4

1
Supported in part by Huagen Pharmaceuticals Company, Ltd. and by the
Ohio Department of Development, Thomas Edison Program.
2
To whom correspondence should be addressed. E-mail: chenx@ohiou.edu.
3
Abbreviations used: BE, banaba hot water extract; BME, banaba HP-20
methanol eluent; BWE, banaba HP-20 water eluent; DEX, dexamethasone;
DMEM, Dulbeccos modified Eagles medium; DPBS, Dulbeccos PBS; FBS, fetal
bovine serum; GLUT4, glucose transporter-4; IBMX, 3-isobutyl-1-methylxanthine;
IS, insulin; KRP, Krebs-Ringer-Hepes; NIDDM, noninsulin-dependent diabetes
mellitus; PPAR2, peroxisome proliferator-activated receptor 2; TZD, thiazolidinedione.

0022-3166/01 $3.00 2001 American Society for Nutritional Sciences.

Manuscript received 12 March 2001. Initial review completed 3 May 2001. Revision accepted 23 June 2001.
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LIPOGENESIS-INHIBITORY AND GLUCOSE UPTAKESTIMULATORY ACTIVITIES OF A PLANT EXTRACT

(GLUT4), a hallmark of adipocyte differentiation and the

molecule that mediates insulin-stimulated glucose transport
(12,13), were studied in 3T3-L1 preadipocytes treated with BE
in the presence or absence of insulin, IBMX or DEX. These
studies were designed to characterize the effects of BE in
3T3-L1 cells at cellular and molecular levels, and to identify
an extract that may be used for prevention and treatment of
obesity and NIDDM without the undesirable side effects of
insulin therapy (19).
MATERIALS AND METHODS

different treatment conditions. To compensate for multiple t tests, P

0.01 was set as the level of significant difference.
Adipocyte differentiation assay. Undifferentiated 3T3-L1 preadipocytes were induced to differentiate into adipocytes as described
above. The degree of the differentiation of the cells induced by
different agents was evaluated by microscopic observation of lipid
accumulation, as well as by the glucose uptake activities they exhibited at the end of the induction as described above. The glucose
uptake assay was chosen and performed here for determination of the
degree of adipocyte differentiation on the basis of the observation
that differentiated adipocytes can be induced by insulin to take up
glucose, whereas preadipocytes cannot (23,24).
Northern blot and Western blot analyses. Total RNA or total
protein was isolated with standard procedures from 3T3-L1 cells
induced by different combinations of insulin, IBMX and DEX, and
BE. For detection of PPAR2 mRNA expression, a 32P-labeled fragment of 308 bp, corresponding to the nucleotides from 29 to 336
of the coding region of the PPAR2 cDNA, was used as a probe and
10 g of total RNA (isolated 144 h postinduction) was used per
sample. For Western blot analysis, an anti-mouse GLUT4 monoclonal antibody was used and 100 g of total protein (isolated 10 d post
induction) was loaded per lane.

RESULTS AND DISCUSSION

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Materials. 3T3-L1 fibroblasts were purchased from American

Type Culture Collection (ATCC, Rockville, MD). Banaba leaves
were obtained as a gift from Huagen Pharmaceuticals (Hong Kong).
Dulbeccos modified Eagles medium (DMEM) and Dulbeccos PBS
(DPBS) were from Gibco Life Technologies (Grand Island, NY).
Fetal bovine serum (FBS) was from Atlanta Biologicals (Norcross,
GA). Bovine IS, IBMX and DEX were from Sigma Chemical (St.
Louis, MO). 2-Deoxy-D-[3H] glucose and XK 50 column were from
Amersham Pharmacia Biotech (Piscataway, NJ). Dianion HP-20
resin was from Mitsubishi Chemical (Tokyo, Japan). Anti-mouse
GLUT4 monoclonal antibody was from Biogenesis (Brentwood,
NH).
Banaba extract preparation. Banaba extract was prepared by the
method described previously (15) with modifications. Banaba hot
water extract (BE) was isolated by boiling 50 g of banaba tea in 1 L
distilled water for 30 min, followed by ultracentrifugation at 30,000
g for 30 min, filtration with a 0.4-m filter, concentration by heat
evaporation and freeze-drying. The BE was further separated by
passage through a Dianion HP-20 resin column. The BE was loaded
on the column packed with Dianion HP-20 resin, then washed with
distilled water (BWE), and the absorbed fraction was eluted with
methanol (BME). These two eluted fractions from the column were
individually concentrated and freeze-dried. The powder of the banaba
extracts (BE and BME) was dissolved in sterile distilled H2O, and
then further sterilized with 0.2-m filters for the adipocyte differentiation study. Unless otherwise stated, BE was used in the study.
Cell culture and adipocyte differentiation. 3T3-L1 cells were
maintained in DMEM and supplemented with 10% FBS at 37C in a
10% CO2 cell incubator. Preadipocyte 3T3-L1 cells were grown in
12-well plates until 2 d postconfluence. The differentiation was
induced as previously described (20) by addition of 1 mg/L IS, 0.5
mmol/L IBMX and 0.25 mol/L DEX (IS-IBMX-DEX). Two days
after induction, the IS-IBMX-DEX containing medium was replaced
with medium containing 1 mg/L IS alone. The medium was subsequently replaced again with fresh culture medium (DMEM supplemented with 10% FBS) after 2 d and then every other day thereafter.
To determine the roles of banaba extract in adipocyte differentiation,
BE was added to the medium either to substitute for insulin (BEIBMX-DEX) or to supplement IS-IBMX-DEX (BE-IS-IBMX-DEX).
The differently induced cells were assayed for glucose uptake activity
9 12 d after the initiation of induction.
Glucose uptake activity assay. Glucose uptake activity was analyzed by measuring the uptake of 2-deoxy-D-[3H] glucose as described
previously (21,22). Briefly, confluent 3T3-L1 adipocytes grown in
12-well plates were washed twice with serum-free DMEM and incubated with 1 mL of the same medium at 37C for 2 h. The cells were
washed 3 times with Krebs-Ringer-Hepes (KRP) buffer and incubated
with 0.9 mL KRP buffer at 37C for 30 min. Insulin and/or BE were
then added and adipocytes were incubated at 37C for 15 min.
Glucose uptake was initiated by the addition of 0.1 mL KRP buffer
and 37 MBq/L 2-deoxy-D-[3H] glucose and 1 mmol/L glucose as final
concentrations. After 10 min, glucose uptake was terminated by
washing the cells 3 times with cold PBS. The cells were lysed with 0.7
mL of 1% Triton X-100 at 37C for 20 min. The radioactivity
retained by the cell lysates was determined by a scintillation counter.
Nonspecific glucose uptake was measured at a glucose concentration
of 100 mmol/L. Assay data were analyzed statistically using Students
t test by comparison of experimental samples of the same treatment
conditions as a group with negative control (untreated) samples, or
positive (insulin-treated) samples, or with experimental samples with

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Although to a lesser extent than insulin, both banaba

extracts (BE and BME) stimulated glucose uptake in 3T3-L1
adipocytes, whereas BWE did not (P 0.01; Fig. 1), suggesting that the effective component(s) with glucose transportinducing activity in the banaba extract are water-soluble but
relatively nonpolar. Also, the maintenance of the activity
through boiling and heat evaporation during extract preparation indicated that the effective component(s) is heat stable
and is unlikely to be a protein(s).
The effect of the concentration of BE on glucose uptake
was compared with that of insulin. The concentration-dependent curve of glucose uptake activity of BE (Fig. 2A) is very
similar to that of insulin (Fig. 2B). The concentration range of
BE that stimulated the greatest glucose uptake was 0.1 0.25
g/L (Fig. 2A). The similarity between the two dose-response
curves and the observation that the induction time required by
BE for stimulating glucose uptake activity was similar to that
of insulin, i.e., no 15 min (refer to the glucose uptake
activity assay in Materials and Methods), suggest that BE may

FIGURE 1 The effect of banaba extract (BE) on glucose uptake in

3T3-L1 adipocytes. Adipocytes in 12-well plates were incubated for 15
min with different BE [hot water extract (BE), HP-20 methanol eluent
(BME) and HP-20 column water eluent (BME)], or with insulin as a
positive control, or without treatment as a negative control, then assayed for 2-deoxy-D-[3H]glucose uptake. Data are means SD, n 8.
Means with different letters differ, P 0.01.

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LIU ET AL.

FIGURE 2 The effect of the concentration of banaba extract (BE,

panel A) or insulin (panel B) on glucose uptake in 3T3-L1 adipocytes.
Differentiated 3T3-L1 cells in 12-well plates were incubated with different concentrations of BE or insulin for 15 min, and then assayed for
2-deoxy-D-[3H]glucose uptake. Data are means SEM, n 6.

stimulate glucose uptake by a mechanism that is similar to that

of insulin (2325) and different from those utilized by other
chemicals (24,26 28).
To test whether BE could further enhance insulins glucose
uptake activity, 0.1 g/L BE was added to insulin at various
concentrations (0 1000 nmol/L). Glucose uptake was not
different from that of insulin alone (Fig. 3A), indicating that
no additive or synergistic effects exist between BE and insulin.
To the contrary, in comparison to insulin alone (the leftmost
data point on the insulin BE curve of Fig. 3B), reduction of
the glucose uptake activity by addition of BE to insulin was
observed (Fig. 3B). On the basis of the observations of glucose
uptakeinducing activity of BE and inhibitory activity of BE
on insulin-induced glucose uptake, a mechanism of action of
BE may be hypothesized. BE may interact with a protein factor
that is directly involved in the insulin-mediated glucose transport signaling pathway that starts with the insulin receptor and
terminates with GLUT4, consequently activating glucose uptake. On the other hand, the interaction between BE and the
protein factor may structurally alter the conformation of the
factor, preventing it from properly receiving the glucose uptake signal initiated from insulin-insulin receptor binding.
Further studies of insulin receptor binding and receptor and
intracellular protein phosphorylation are needed for the final
elucidation of the site of action and the glucose uptake induction mechanism mediated by BE. BE apparently stimulates
glucose uptake through a mechanism that is very different
from that used by other common antidiabetic drugs such as the
thiazolidinediones (TZD) (29,30). TZD stimulate glucose up-

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take using a slow and indirect mechanism by activating

PPAR, which in turn up-regulates GLUT4 gene expression
(29 31). In contrast, the glucose uptakestimulatory activity
of BE seems to be much faster and more direct. The effective
compound(s) in BE may represent a new group of chemicals
that could be used as alternatives to TZD to induce glucose
uptake in both cells and animals.
Undifferentiated 3T3-L1 preadipocytes can be converted to
adipocytes by addition of a cocktail containing insulin, IBMX
and DEX (20). However, when 1100 mg/L BE was substituted
for insulin and added to preadipocytes in the presence of
IBMX and DEX, no adipocyte differentiation of 3T3-L1 cells
was observed as revealed by glucose uptake assays (Fig. 4).
This result indicates that BE does not induce adipocyte differentiation in 3T3-L1 cells. Interestingly, both BE and BME,
when co-incubated with IS-IBMX-DEX, inhibited adipogenesis, whereas BWE did not (Fig. 5). This indicates that both the
adipocyte differentiation inhibition activity and the glucose
uptake activity were due to BME (Fig. 1). The inhibition of
adipocyte differentiation by BE was time (Fig. 6A) and concentration dependent (Fig. 6B). These results are consistent
with the microscopic observations of fat accumulation (Fig. 5).
The 3T3-L1 preadipocytes, whose differentiation was blocked
by co-incubation of BE and IS-IBMX-DEX, retained the capacity to reenter the differentiation process when the ISIBMX -DEX induction cocktail was reintroduced into the cells
(data not shown). It is interesting to note that BE showed an

FIGURE 3 Combined effects of insulin and banaba extract (BE) on

glucose uptake in 3T3-L1 adipocytes. Differentiated 3T3-L1 cells were
incubated with insulin in the presence or absence of 0.1 g/L BE (A), or
incubated with BE in the presence or absence of 1 mol/L insulin (B) for
15 min, and then assayed for the glucose uptake activities. Data are
means SEM, n 6.

LIPOGENESIS-INHIBITORY AND GLUCOSE UPTAKESTIMULATORY ACTIVITIES OF A PLANT EXTRACT

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FIGURE 4 The effect of banaba extract (BE) on adipocyte differentiation in the absence of insulin in 3T3-L1 cells. Undifferentiated
3T3-L1 preadipocytes were induced by either insulin or BE in the
presence of dexamethasone (DEX) and 3-isobutyl-1-methylxanthine
(IBMX). Ten days after induction, the degree of adipocyte differentiation
was assayed by the glucose uptake activities of the cells. Data are
means SD, n 6. Means with different letters differ, P 0.01.
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FIGURE 6 The effects of introduction time and concentration of

banaba extract (BE) on adipocyte differentiation in the presence of
insulin (IS) in 3T3-L1 cells. Preadipocyte 3T3-L1 cells were induced with
IS in the presence of dexamethasone (DEX) and 3-isobutyl-1-methylxanthine (IBMX) at d 0. BE was added to the media either at various
times after the initial induction (A) or at various concentrations at the
time of the initial induction (B). The degree of differentiation of differently treated 3T3-L1 cells was assayed by the glucose uptake activities
of the cells. Data are means SD, n 6. Means with different letters
differ, P 0.01.

FIGURE 5 The effect of different banaba extract (BE) on adipocyte differentiation in the presence of insulin (IS) in 3T3-L1 cells. In the
presence of dexamethasone (DEX) and 3-isobutyl-1-methylxanthine
(IBMX), undifferentiated 3T3-L1 preadipocytes were induced by either
insulin, (BE IS), (BME IS) or (BWE IS). Ten days after the
induction, the cells were photographed at magnification X200. (A) No
induction; (B) IS; (C) BE (0.5 g/L) and IS; (D) BME (0.5 g/L) and IS; (E)
BWE (0.5 g/L) and IS. Figure shown represents one of four independent
experiments. All four experiments showed similar results. Abbreviations: BME, banaba HP-20 methanol eluent; BWE, banaba HP-20 water
eluent.

insulin-like glucose uptakeinducing activity in adipocytes but

did not show an insulin-like differentiation-inducing activity
in preadipocytes. This difference may be explained by the facts
that these two activities involve two distinct signaling pathways and that the insulin receptor is involved in glucose
transport in adipocytes, whereas the insulin-like growth factor
1 receptor, which is homologous to the insulin receptor, is used
by insulin in preadipocytes for induction of differentiation
(18,32,33).
To investigate how BE inhibits adipocyte differentiation
induced by IN-IBMX-DEX, two important differentiation
markers, PPAR2 and GLUT4, were used to monitor the
progress of differentiation in the preadipocytes that were induced by either IBMX-DEX or IS-IBMX-DEX in the presence
or absence of BE. Northern blot analysis revealed that BE
greatly inhibited the mRNA expression of PPAR2 induced

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LIU ET AL.

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observed in undifferentiated cells (Fig. 7B). GLUT4 expression was used as an indicator of differentiation in this study to
monitor the ability of BE to induce differentiation in preadipocytes, not glucose transport in adipocytes. All of these
results suggest that the specific target of the differentiation
inhibition exerted by BE is PPAR2 or factor(s) that directly
or indirectly regulate the expression of PPAR2. Further experiments are necessary to determine the site of the inhibition.
The identity and function of the 43-kDa protein (Fig. 7B) are
not known at this time. The presence of such a protein band
in the Western blot was likely due to the specific monoclonal
antibody, and possibly the cells used in this study.
It is also interesting and important to determine whether
the glucose uptake and adipocyte differentiation activities are
mediated by the same or different compounds in BE. We used
HPLC to analyze and isolate the effective compound(s) from
BE. Up to now, the differentiation-inhibitory activity was
always associated with the glucose uptakeinducing activity in
a single fraction or peak isolated by HPLC (data not shown).
These observations strongly suggest that the two activities
come from the same compound in BE. The final isolation and
characterization of the effective compound(s) are in progress.
We have demonstrated that BE stimulates glucose uptake
activity and inhibits the adipocyte differentiation activity of
IS-IBMX-DEX in 3T3-L1 cells. These new findings are consistent with the previous observations that BE lowered blood
glucose levels in diabetic mice (15) and reduced weight gain
and adipose tissue mass in female diabetic mice (16).
Antidiabetic drugs such as insulin or TZD up-regulate both
glucose transport and lipid biosynthesis in adipocytes (29,30).
Weight gain is a frequent side effect of insulin therapy in type
II diabetic patients (19). Therefore, drugs with glucose-lowering activity, but lacking adipogenic activity are highly desirable. The effective component(s) of BE seem to have such an
advantageous combination. A new polypeptide hormone, resistin, has recently been found in adipocytes to be one of the
potential links between obesity and type II diabetes (34,35).
Resistin may be responsible for insulin resistance, and its gene
expression profile appears to be very similar to that of PPAR
(34), a gene that BE down-regulates (Fig. 7A). Thus, an
understanding of the mechanism of BE action will be valuable
for the study, prevention, and treatment of obesity, insulin
resistance and type II diabetes.

FIGURE 7 Expression of peroxisome proliferator-activated receptor 2 (PPAR2) or glucose transporter 4 (GLUT4) in 3T3-L1 cells
induced under different conditions. Preadipocytes were induced with or
without insulin in the presence or absence of banaba extract (BE). Total
RNA or protein was isolated from the differently treated cells and
analyzed for the expression of PPAR2 mRNA and GLUT4 protein. (A)
Northern blot analysis of PPAR2 expression in 3T3-L1 cells 144 h
postinduction; 10 g of total RNA was used per lane. (B) Western blot
analysis of GLUT4 expression in 3T3-L1 cells 10 d post induction; 100
g of total protein was used per lane. GLUT4 has a molecular mass of
65 kDa. Figure shown here represents one of three independent
experiments. All three experiments showed similar results.

by either IBMX-DEX or IS-IBMX-DEX in a dose-dependent

manner (Fig. 7A). Furthermore, as shown by a Western blot
analysis, the protein production of GLUT4 (65 kDa) was
inhibited more in the cells induced by IS-IBMX-DEX in the
presence of BE than in the cells induced in the absence of BE
(Fig. 7B). These results are consistent with our other differentiation inhibition results studied with glucose uptake assays
(Figs. 4 and 6), and are not inconsistent with other glucose
uptake assays (Figs. 13) because the glucose uptakeinducing
activity of BE was examined in fully differentiated adipocytes
(Figs. 13), whereas the GLUT4 inhibitory effect of BE was

ACKNOWLEDGMENTS
We thank J. Butcher and L. Lapierre for critical review and
comments on the manuscript, and K. Walker for technical assistance.

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